Genotyping minisequencing

Figure 1 In the array-of-arrays conformation a standard microscope slide is divided into 80 subarrays with a diameter identical to that of a 384-well-microtiter-plate reaction well, schematically illustrated to the left in the image. Up to 200 oligonucleotide spots can be printed per subarray at a center-to-center distance of 200 pm. With the possibility to analyze 80 samples in parallel, up to 16,000 genotypes per slide can be generated. Fluorescence images of four reaction wells, where the minisequencing reaction have been performed are shown to the right.

Figure 2 Minisequencing reaction on with extension primers immobilized on the microarray surface. The multiplex PCR products of the regions containing the SNPs are allowed to hybridize to the oligonucleotides immobilized on the microarray (A). The primers are extended with fluorescently labelled ddNTPs complementary to the nucleotide at the SNP position. Four different fluorophores, one for each nucleotide is used allowing for a simultaneous detection of the four nucleotides in one single reaction (B). After washing the slide with sodium hydroxide and salt buffers, only the extended primers covalently attached to the surface remains and the fluorescence is measured (C) and the genotypes assigned.

Lovmar L, Fredriksson M, Liljedahl U, Sigurdsson S, Syvanen AC. Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA. Nucleic Acids Res 2003 31 el29. 

Lovmar, L. and A. Syvanen, Genotyping single-nucleotide polymorphisms by minisequencing using tag arrays. Methods in Molecular Medicin, 2005.114 p. 79-92. 

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